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15-PGDH inhibition in multiple models of murine BMT
Introduction
Hematopoietic stem cells (HSC) are rare, primitive cells in the blood and bone marrow which give rise to all dif- ferentiated blood cells.1-6 HSC are transplanted therapeuti- cally after myeloablative chemotherapy as part of poten- tially curative treatment regimes for a variety of malignant and non-malignant disorders.7-10 However, profound neu- tropenia during the period while awaiting hematopoietic recovery after hematopoietic stem cell transplantation (HSCT) results in a high risk of opportunistic infection, which is a major source of morbidity, mortality, and pro- longed hospitalization associated with the procedure.11,12 Strategies to accelerate hematopoietic regeneration fol- lowing HSCT are thus of therapeutic interest. The eicosanoid signaling molecule prostaglandin E2 (PGE2), synthesized by the cyclo-oxygenase isoenzymes, has been recognized by several investigators as of interest for supporting hematopoietic regeneration. Ex vivo incubation of whole bone marrow with PGE2 has been shown to enhance HSC homing and proliferation in murine and non-human primate transplant models, and non-random- ized clinical studies suggested potential for similar benefit in humans.13-17 In addition, in vivo treatment with PGE2 shows protective effects on murine hematopoietic cell populations following sublethal radiation via upregulation of cellular survival pathways.18,19 These observations sug- gest that in vivo modulation of PGE2 signaling could also potentiate hematopoietic recovery following HSCT.
We have previously identified 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme that mediates the first and rate-limiting step in PGE2 degradation in vivo, as a therapeutic target that, by acting on the bone marrow HSC niche, can potentiate hematopoietic recovery after HSCT.20 In particular, we described the discovery of SW033291, a potent small molecule 15-PGDH inhibitor (Ki = 0.1 nM).20 In in vivo studies, we have shown that, by inhibiting 15-PGDH, SW033291 doubles bone marrow PGE2 levels, induces bone marrow stromal production of CXCL12 and stem cell factor, potentiates HSC homing to the bone marrow niche, potentiates bone marrow colony- forming capacity, and, in murine HSCT, accelerates recov- ery of neutrophil counts by 6 days, enhances survival, and shows no long-term effects on serial transplantation capacity.20 In subsequent medicinal chemistry studies we have now developed SW209415, a second-generation 15- PGDH inhibitor, which has a 10,000-fold enhanced aque- ous solubility of 4300 mg/mL (as an HCl salt) as compared to SW033921.21 This improved aqueous solubility of SW209415 would enable administration in a human- acceptable intravenous formulation.21 SW209415 modifies SW033291 by substituting a dimethyl-imidazole in place of a previous phenyl group and substituting a thiazole ring in place of a prior thiophene (Online Supplementary Figure S1). Moreover, we identified that all of the 15-PGDH inhibitory activity of both SW033291 and SW209415 lies in their respective (+)-enantiomers.21 We herein now describe the in vivo biological activities of (+)-SW209415 in modulating tissue PGE2, and in enhancing recovery from HSCT in an expanded range of biological models relevant to human HSCT, and furthermore evaluate concerns that 15-PGDH inhibitors, by increasing PGE2, could potentiate the in vivo growth of cancers. These studies provide posi-
tive findings that advance 15-PGDH as a therapeutic tar- get for potentiating HSCT.
Methods
Bone marrow homing
Bone marrow homing to the recipient niche was measured by labeling donor marrow with 5 mM CellTrace CFSE (Life Technologies) for 30 min at 37°C and transplanting 10x106 labeled cells into lethally irradiated recipient mice (of the same age, gender, and strain). Recipient mice were give three intraperitoneal injec- tions of vehicle or 5 mg/kg (+)-SW209415.
Colony-forming unit – spleen assay
Eight-week old C57BL/6J mice were lethally irradiated with 11 Gy and transplanted with 200x103 total bone marrow cells. Recipients were treated intraperitoneally with either vehicle or 2.5 mg/kg (+)-SW209415 twice daily for 12 days. On day 12 mice were sacrificed and spleens harvested and assessed for colony counts and SKL determination.
Bone marrow transplantation
Eight-week old female C57BL/6J mice were lethally irradiated with 11 Gy and transplanted with 500x103 total bone marrow cells 16 h after irradiation. Recipients were treated intraperitoneally with either vehicle or 2.5 mg/kg (+)-SW209415 twice daily through the course of the experiment. In studies involving granu- locyte colony-stimulating factor (G-CSF), cohorts of mice were additionally treated once daily subcutaneously with 250 mg/kg human G-CSF or the combination of human G-CSF and (+)- SW209415.
Human bone marrow and umbilical cord blood studies
Discarded, de-identified human umbilical cord blood (3 unique samples) and adult bone marrow aspirates [2 unique samples from a 28-year old male (28/M) and a 50-year old male (50/M)] were obtained from the Case Western Reserve University Hematopoietic Biorepository with permission from the Institutional Review Board. Buffy coat from umbilical cord sam- ples and total marrow from aspirates were incubated with car- boxyfluorescein succinimidyl ester (CFSE) and the homing assay performed as described above. Engraftment at 12 weeks was stud- ied by transplanting 1x106 mononuclear cells into each recipient NSG mouse. This was performed using two adult bone marrow aspirates (37 M and 41 M). Mice were treated twice daily for 21 days after the transplant with vehicle or 2.5 mg/kg (+)-SW209415 and bled at serial time-points through to day 84. Peripheral blood multilineage differentiation was assessed via flow cytometry (using CD3, B220, and CD11b gated from human CD45 cells).
Xenograft studies
In vivo growth of human acute myeloid leukemia (AML) and human multiple myeloma (MM) was established by transplanting 5x106 total human AML cells (cultured from a human AML patient at University Hospitals) or MM cells (ATCC line RPMI 8226), via the tail vein, into NSG mice receiving 2.5 Gy irradiation. Treatment was initiated 2 weeks after transplantation, with mice then started on twice daily intraperitoneal injections with either vehicle or 2.5 mg/kg (+)-SW209415 until the animals began to demonstrate noticeable signs of hunching and lethargy, at which point they were sacrificed to assess human CD45+ cells in the bone marrow of those that had received AML cells and human CD38+ cells in the bone marrow of those that had received MM cells.
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